title: Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy.
abstract: The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD- or ETD-based tandem mass spectrometry alone.
authors.affiliation: Michael Barber Centre for Mass Spectrometry, School of Chemistry and Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom.
authors.forename: Sarah R
authors.initials: SR
authors.lastname: Hart
authors.forename: King Wai
authors.initials: KW
authors.lastname: Lau
authors.forename: Zhiqi
authors.initials: Z
authors.lastname: Hao
authors.forename: Richard
authors.initials: R
authors.lastname: Broadhead
authors.forename: Neil
authors.initials: N
authors.lastname: Portman
authors.forename: Andreas
authors.initials: A
authors.lastname: Hühmer
authors.forename: Keith
authors.initials: K
authors.lastname: Gull
authors.forename: Paul G
authors.initials: PG
authors.lastname: McKean
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Hubbard
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Gaskell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005407:Flagella / Q000737:chemistry*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014345:Trypanosoma / Q000737:chemistry*
chemical_list: D020543:Proteome